Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Evidence for bidirectional endocannabinoid transport across cell membranes

Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as 2-arachidonoylglycerol (2-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and 2-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nm AEA and 1 μm 2-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and 2-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 μm) and OMDM-2 (5 μm), a functional synergism on cellular AEA and 2-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and 2-AG. We show, for the first time, that UCM707 and OMDM-2 inhibit the bidirectional movement of AEA and 2-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and 2-AG trafficking and metabolism.

Annexins sense changes in intracellular pH during hypoxia

The pH(i) (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pH(i). Sensors of pH(i) include ion transport systems which control intracellular Ca2+ gradients and link changes in pH(i) to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca(2+)-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2-S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pH(i) on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2-S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2-S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2-S100A10 might have consequences for their functions as membrane organizers and channel modulators.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Effect of systemic tetracycline on the degradation of tetracycline-impregnated bilayered collagen membranes: an animal study

BACKGROUND: Premature collagen membrane degradation may compromise the outcome of osseous regenerative procedures. Tetracyclines (TTCs) inhibit the catalytic activities of human metalloproteinases. Preprocedural immersion of collagen membranes in TTC and systemic administration of TTC may be possible alternatives to reduce the biodegradation of native collagen membranes. AIM: To evaluate the in vivo degradation of collagen membranes treated by combined TTC immersion and systemic administration. MATERIALS AND METHODS: Seventy-eight bilayered porcine collagen membrane disks were divided into three groups and were immersed in 0, 50, or 100 mg/mL TTC solution. Three disks, one of each of the three groups, were implanted on the calvaria of each of 26 Wistar rats. Thirteen (study group) were administered with systemic TTC (10 mg/kg), while the remaining 13 received saline injections (control group). Calvarial tissues were retrieved after 3 weeks, and histological sections were analyzed by image analysis software. RESULTS: Percentage of remaining collagen area within nonimpregnated membranes was 52.26 ± 20.67% in the study group, and 32.74 ± 13.81% in the control group. Immersion of membranes in 100 mg/mL TTC increased the amount of residual collagen to 63.46 ± 18.19% and 42.82 ± 12.99% (study and control groups, respectively). Immersion in 50 mg/mL TTC yielded maximal residual collagen values: 80.75 ± 14.86% and 59.15 ± 8.01% (study and control groups, respectively). Differences between the TTC concentrations, and between the control and the study groups were statistically significant. CONCLUSIONS: Immersion of collagen membranes in TTC solution prior to their implantation and systemic administration of TTC significantly decreased the membranes' degradation.

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-gamma and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-gamma and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli.

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells

http://www.ncbi.nlm.nih.gov/pubmed/20381326

Different collagen types define two types of idiopathic epiretinal membranes

To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type.

The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.

Plasma membrane repair and cellular damage control: the annexin survival kit

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury.